Biology of a New DAP12 Associated Receptor Family

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DESCRIPTION (provided by the applicant): By express cloning, we have identified a new family of mouse receptors that associate with the signaling accessory molecule, DAP12. They have a single Ig-like domain, and their expression appears to be restricted to cells of monocyte/macrophage lineage. Thus, we have named them "MDI receptors" (for myeloid cell DAP12-associated Ig-like receptors). We have identified 3 overlapping BACs containing MDI genes and have mapped the genes to chromosome 17d. Digests of the BACs indicate that the MDI receptor family is small, probably limited to two homologous genes, MDI-1 and MDI-2. When expressed in MT2 macrophages, MDI-1 associates with endogenous DAP12 and stimulates the production of nitric oxide. Only 3 other receptors are known to associate with DAP12 in monocyte/macrophage cells. Mice deficient in Dap12 have immune alterations that appear to lie in antigen presenting cells, and humans deficient in DAP12 have neurologic and bone disorders that may reflect defects in microglial cells and osteoclasts, respectively. We therefore wish to define the functions of the MDI receptors and their ligands. We propose five specific aims: Specific Aim 1. Define the ligands for MDI-1 and MDI-2. We will create soluble MDI-1 and MDI-2 receptors to identify ligands on other cells or, if indicated, from serum or other sources, We will seek to identify the ligands either as known proteins or, if they are unknown, to clone the cDNA(s) for the ligand(s). Specific Aim 2. Define the extent of the MDI receptor family. We will further probe monocyte/macrophage cDNA libraries to seek additional members of the MDI family. Second we will map and sequence the MDI genes that are encoded in the three BACs that hybridize to MDI transcripts. Specific Aim 3. Define the range of expression of MDI receptors. We will produce monoclonal antibodies against individual MDI receptors, and we will use these to assess the surface expression of MDI on different cell types and at different stages of cell activation. Specific Aim 4. Define the cellular responses to ligation of MDI. We will study both receptor-transfected cells and freshly prepared cells. Our studies will be guided by our own results and by the phenotype of DAP12-deficient mice and humans. They include a collaboration with Dr. Jason Cyster regarding chemokines and chemokine receptors. Specific Aim 5. Define the phenotype of MDI-/- mice. In collaboration with Nigel Killeen, we will create mice deficient in both MDI-1 and MDI-2. Again, these studies will be guided by our own results as well as the phenotype of DAP12-deficient mice and humans.